Note: Descriptions are shown in the official language in which they were submitted.
CA 02379022 2002-O1-11
WO 01/05810 PCT/EP00/06188
Cyclic peptide derivatives as inhibitors of the
integrin pc"~6
The invention relates to novel peptide derivatives of
the formula I
cyclo (Arg-X1-Asp-X2-X3-X4-XS-X6-R1) I
in which
X1 is Ser, Gly or Thr,
X2 is Leu, Ile, Nle, Val or Phe,
X3 is Asp, Glu, Lys or Phe,
X4 is Gly, Ala or Ser,
XS is Leu, Ile, Nle, Val or Phe,
X6 is Arg, Har or Lys,
R1 is absent
or is
one or
more
w-aminocarboxylic
acid
residue(s), the cu-aminocarboxylic acid residues)
having a
length
of
500
to
2500
pm,
where the amino acids mentioned can also be
derivatized,
the D and the L forms of the optically active amino
acid residues are included,
and their physiologically acceptable salts and
solvates.
The invention was based on the obj ect of finding novel
compounds having valuable properties, in particular
those which can be used for the production of
medicaments.
It has been found that the compounds according to the
invention and their salts have very valuable
pharmacological properties together with good
tolerability.
The peptides according to the invention can be employed
as efficacious inhibitors of the 0c"(36 integrin receptor
CA 02379022 2002-O1-11
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and thus for the treatment of various diseases and
pathological findings.
Other inhibitors of the integrin a,"~36 are described in
DE 19858857 and by S. Kraft et al. in J. Biol. Chem.
274, 1979-85 (1999). The compounds according to the
invention are to be considered as a selection invention
with respect to the application mentioned.
Integrins belong to the family of heterodimers of Class
I - transmembrane receptors which play an important
role in numerous cell-matrix or cell-cell adhesion
processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol.
47). They can be roughly divided into three classes:
the (31 integrins, which are receptors for the
extracellular matrix, the (32 integrins, which are
activatable on leucocytes and are "triggered" during
inflammatory processes, and the oc~, integrins, which
influence the cell response during wound-healing and
other pathological processes (Marshall and Hart, 1996,
Semin. Cancer Biol. 7, 191).
The integrins OcS~il , oclibø3 , Cca~l , oc,"(~1, oc~,(33 , oc~,~is , oc~,(38
and
oc"(36 all bind to the Arg-Gly-Asp (RGD) peptide sequence
in natural ligands, such as, for example, fibronectin
or vitronectin. Soluble RGD-containing peptides are
able to inhibit the interaction of each of these
integrins with the corresponding natural ligands. an,(36
is a relatively rare integrin (Busk et al., 1992 J.
Biol. Chem. 267(9), 5790), which is formed to an
increased extent in repair processes in epithelial
tissue and preferably binds the natural matrix
molecules fibronectin and tenascin (Wang et al., 1996,
Am. J. Respir. Cell Mol. Biol. 15(5), 664). The
physiological and pathological functions of 0c"(36 are
still not precisely known; it is suspected, however,
that this integrin plays an important role in
physiological processes and disorders (e. g.
inflammation, wound healing, tumours) in which
CA 02379022 2002-O1-11
- 3 -
epithelial cells are involved. Thus o~,(36 is expressed on
keratinocytes in wounds (Haapasalmi et al., 1996, J.
Invest. Dermatol. 106(1), 42), from which it is to be
assumed that in addition to wound-healing processes and
inflammation, other pathological skin events, such as,
for example, psoriasis, can also be influenced by
agonists or antagonists of the said integrin. oc~,(36
furthermore plays a role in the respiratory tract
epithelium (Weinacker et al., 1995, Am. J. Respir. Cell
Mol. Biol. 12(5), 547), so that appropriate
agonists/antagonists of this integrin could. be
successfully employed in respiratory tract disorders,
such as bronchitis, asthma, pulmonary fibrosis and
respiratory tract tumours. Finally, it is known that
oc~,~36 also plays a role in the intestinal epithelium, so
that appropriate integrin agonists/antagonists could be
used in the treatment of inflammation, tumours and
wounds of the stomach/intestinal tract.
The dependence of the formation of angiogenesis on the
interaction between vascular integrins and extra-
cellular matrix proteins is described by P.C. Brooks,
R.A. Clark and D.A. Cheresh in Science 264, 569-71
(1994).
The object was therefore, in addition to the previously
known natural high molecular weight ligands and
antibodies, which are therapeutically and diagnos-
tically difficult to handle, to find potent, specific
and selective low molecular weight ligands for oc~,(36,
preferably peptides, which can be used for the
therapeutic areas mentioned, but also as diagnostic or
reagent.
It has been found that the peptide compounds according
to the invention and their salts, as soluble molecules,
exert an effect on cells which carry the said receptor,
or, if they are bound to surfaces, are artificial
ligands for oc,~,(36-mediated cell adhesion. Above all, they
CA 02379022 2002-O1-11
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act as oc,~,(36 integrin inhibitors, where they particularly
inhibit the interactions of the receptor with other
ligands, such as, for example, the binding of fibro-
nectin. This action can be detected, for example, by
the method which is described by J.W. Smith et al. in
J. Biol. Chem. 265, 12267-12271 (1990).
It has furthermore been found that the novel substances
have very valuable pharmacological properties together
with good tolerability and can be employed as
medicaments. This is described more precisely further
below.
The peptide compounds according to the invention can
furthermore be used in vivo and in vitro as diagnostics
for the detection and localization of pathological
conditions in the epithelial system, if they are
equipped with appropriate markers (e. g. the biotinyl
radical) according to the prior art.
The invention also comprises combinations with at least
one other active compound and/or conjugates with other
active compounds, such as cytotoxic active compounds
and conjugates with radiolabels for X-ray therapy or
PET diagnosis, but also fusion proteins with marker
proteins such as GFP or antibodies, or therapeutic
proteins such as IL-2.
Particularly active compounds are those of the formula
I in which an octapeptide sequence cyclo (Arg-X1-Asp-X2-
X3-X9-XS-X6) , in which the radicals Xl, X2, X3, X4, XS and
X6 have the meanings indicated, is expanded by Rl. The
effect of the ring expansion is shown in Fig. 1 by the
example of cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg)
[EMD 271588]. Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NHZ
[E1~ 271293] serves as a comparison compound.
Some cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-R1)
peptides, the distance from R1 (calculated) and the
value Q (ICSO[substance]/ICSO[EI~ 271293]) and -log Q
are given in the following table.
CA 02379022 2002-O1-11
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R1 Distance [pm] Q -log Q
0 0 47 -1.672
Gly 370 2.1 -0.322
Abu 617 0.028 1.553
Gly-Gly 740 0.05 1.301
~a g70 0.036 1.444
Aee 1078 0.038 1.420
Gly-Gly-Gly 1110 0.03 1.523
Abu-Abu 1235 0.03 1.523
Gly-Gly-Gly-Gly 1480 0.033 1.481
Aha-Aha 1740 0.036 1.444
Gly-Gly-Gly-Gly-Gly 1850 0.046 1.337
Gly-(Gly)4-Gly 2220 0.05 1.301
The graphic representation can be seen in Fig. 1.
Particularly active compounds are obtained even if the
spacer length R1 has reached approximately 500 pm.
Particularly preferred compounds of the formula I are
those in which R1 is one or more co-aminocarboxylic acid
residue(s), the c~-aminocarboxylic acid residues)
having a length of 600 to 2500 pm, very particularly
preferably those having a spacer length of 600 to
2000 pm.
c~.~-Aminocarboxylic acid residues) is/are understood as
meaning any c~-aminocarboxylic acid, among which the
amino acids below selected from the group consisting of
Ala, Asn, Asp, Arg, Cys, Gln, Glu, Hcy, His, Hse, Ile,
Leu, Lys, Met, Pen, Phe, Pro, Ser, Thr, Trp, Tyr, Val
arid HZN- ( CH2CH20 ) m- ( CH2 ) n-COOH ,
where m, n in each case independently of one
another are 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11 or 12,
with the proviso that m + n is > 0,
are particularly preferred.
CA 02379022 2002-O1-11
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The invention thus preferably relates to peptide
derivatives according to Claim 1, of the formula I
Cyclo (Arg-X1-Asp-Xz-X3-X4-XS-X6-R1) I
in which
X1 is Ser, Gly or Thr,
X2 is Leu, Ile, Nle, Val or Phe,
X3 is Asp, Glu, Lys or Phe,
X4 is Gly, Ala or Ser,
XS is Leu, Ile, Nle, Val or Phe,
X6 is Arg, Har or Lys,
R1 is absent or is 1-10 amino acids
selected from the
group consisting of Al:a, Asn, Asp, Arg, Cys, Gln,
Glu, Hcy, His, Hse, Ile, Leu, Lys, Met, Pen, Phe,
Pro, Ser, Thr, Trp, Tyr, Val
and
H2N- ( CH2CH20 ) m- ( CHZ ) r-COOH,
m,n in each case independently of one another are 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12,
with the proviso that m + n is > 0,
where the amino acids mentioned can also be
derivatized,
the D and the L forms of the optically active amino
acid residues are included,
and their physiologically acceptable salts.
Some preferred groups of compounds can be expressed by
the following subformulae Ia to Ih, which correspond to
the formula I and in which the radicals not designated
in greater detail have the meaning [sic] indicated in
the formula I, but in which
in a) X1 is Gly or Thr;
in b) X1 is Gly or Thr,
XZ is Leu;
CA 02379022 2002-O1-11
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in c) X1 is Gly or Thr,
XZ is Leu,
X3 is Asp or D-Asp;
in d) X1 is Gly or Thr,
X2 i s Leu ,
X3 is Asp or D-Asp,
X4 is Gly or Ala;
in e) X1 is Gly or Thr,
XZ is Leu,
X3 is Asp or D-Asp,
X4 is Gly or Ala,
XS is Leu;
in f) X1 is Gly or Thr,
Xz is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser,
XS is Leu,
X6 is Arg;
in g) X1 is Gly or Thr,
Xz is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser,
XS is Leu,
X6 i s Arg ;
R1 is 1-10 amino acids selected from the
group consisting of Ala, Asn, Asp, Arg,
Cys, Gln, Glu, Hcy, His, Hse, Ile, Leu,
Lys, Met, Pen, Phe, Pro, Ser, Thr, Trp,
Tyr , Val and H2N- ( CHzCH20 n-COOH,
) m- ( CH2 )
m,n in each case independently of one
another are 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11 or 12,
with the proviso that m + n is > 0;
CA 02379022 2002-O1-11
-
in h) X1 is Gly or Thr,
X2 is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser,
XS is Leu,
X6 is Arg;
R1 is 1-6 amino acids selected from the
group consisting of Gly, (3-Ala, Abu or
Aha;
and their salts.
The invention relates in particular to peptide
compounds selected from the group consisting of
cyclo(Arg-Gly-Asp-Leu-Asp-Ala-Leu-Arg-Gly-Gly-Gly),
cyclo(Arg-Gly-Asp-Leu-Asp-Gly-Leu-Arg-Gly-Gly-Gly),
cyclo(Arg-Gly-Asp-Leu-D-Ala-Ala-Leu-Arg-Gly-Gly-Gly),
cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Gly-Gly-Gly),
cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-Abu),
cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-Aha-Aha),
cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-Aha),
cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-Aee),
cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-Abu),
cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-~3-Ala),
cyclo(Arg-Gly-Asp-Leu-D-Asp-Ala-Leu-Arg-~-Ala),
and their physiologically acceptable salts.
The abbreviations of amino acid residues mentioned
above and below stand for the radicals of the following
amino acids:
Abu 4-aminobutyric acid
3 5 Aee HZN- ( CHZCH20 ) 2-CHZCOOH
Aha 6-aminohexanoic acid, 6-aminocaproic acid
Aib a.-aminoisobutyric acid
Ala alanine
Asn asparagine
CA 02379022 2002-O1-11
_ g _
Asp aspartic acid
Arg arginine
Bgl C-alpha-tert-butylglycine
Cys cysteine
Dab 2,4-diaminobutyric acid
Dap 2,3-diaminopropionic acid
Gln glutamine
Glp pyroglutamic acid
Glu glutamic acid
Gly glycine
Har homoarginine
Hcy homocysteine
His histidine
homo-Phe homophenylalanine
Hse homoserine
Ile isoleucine
Leu leucine
Lys lysine
Met methionine
Nal naphth-2-ylalanine
Nle norleucine
Orn ornithine
Pen penicillamine
Phe phenylalanine
Phg phenylglycine
4-Hal-Phe 4-halophenylalanine
Pro proline
Ser serine
Thr threonine
TIS triisopropylsilane [sic]
Trp tryptophan
Tyr tyrosine
Val valine.
In addition, the following have the meanings below:
Ac acetyl
BOC tert-butoxycarbonyl
BSA bovine serum albumin
CA 02379022 2002-O1-11
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CBZ or benzyloxycarbonyl
Z
DCC1 dicyclohexylcarbodiimide
DCM dichloromethane
DIEA diethylamine
DMF dimethylformamide
EDC1 N-ethyl-N, N'-(dimethylaminopropyl)-
carbodiimide
Et ethyl
FCA fluoresceincarboxylic acid
FITC fluorescein isothiocyanate
Fmoc 9-fluorenylmethoxycarbonyl
FTH fluoresceinthiourea
HOBt 1-hydroxybenzotriazole
Me methyl
MBHA 4-methylbenzhydrylamine
Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl
HONSu N-hydroxysuccinimide
OBut tert-butyl ester
Oct octanoyl
OMe methyl ester
OEt ethyl ester
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
POA phenoxyacetyl
Sal salicyloyl
TBS++ tris buffered saline with divalent cations
TBSA TBS + BSA
TBTU 2-(1H-benzotriazol-1-yl)-1,1,3-
tetramethyluronium tetrafluoroborate
TFA trifluoroacetic acid
Trt trityl (triphenylmethyl).
If the abovementioned amino acids can occur in two or
more enantiomeric forms, all these forms and their
mixtures (for example the DL forms) are included above
and below. In addition, the amino acids can be provided
with appropriate protective groups which are known per
se.
CA 02379022 2002-O1-11
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The invention also relates to the hydrates and
solvates, e.g. alcoholates, of these compounds.
So-called prodrug derivatives are included in the
compounds according to the invention, that is compounds
modified with, for example, alkyl or acyl groups,
sugars or oligopeptides, which are cleaved rapidly in
the body to give the active compounds according to the
invention. These also include biodegradable polymer
derivatives of the compounds according to the
invention, as is described, for example, in Int. J.
Pharm. 115, 61-67 (1995).
The amino acids and amino acid residues mentioned, such
as, for example, the NH functions or terminal amide
functions, can also be derivatized, the N-methyl, N-
ethyl, N-propyl, N-benzyl or Ca-methyl derivatives
being preferred. Derivatives which are additionally
preferred are those of Asp and Glu, in particular the
methyl, ethyl, propyl, butyl, tert-butyl, neopentyl or
benzyl esters of the side chain carboxyl groups, and in
addition also derivatives of Arg, which can be
substituted on the -NH-C(=NH)-NHz group by an acetyl,
benzoyl, methoxycarbonyl or ethoxycarbonyl radical.
In the compounds according to the invention, which are
linked to one another in peptide fashion via the
oc-amino and oc-carboxy groups (head-tail linkage) , those
cyclic compounds are also contained which, e.g. in the
presence of a functional side chain, such as, for
example, an SH group, are linked in the following way
side-side e.g. S-S (disulfide),
head-side
side-tail.
Furthermore, derivatives additionally included in the
compounds according to the invention are those which
CA 02379022 2002-O1-11
- 12 -
consist of the actual peptides according to the
invention and known marker compounds which make it
possible to detect the peptides easily. Examples of
such derivatives are radiolabelled, biotinylated or
fluorescence-labelled peptides.
A fluorescent dye radical is preferably 7-acetoxy-
coumarin-3-yl, fluorescei~-5-(and/or 6-)yl, 2',7'-
dichlorofluorescein-5-(and 6-)yl, dihydrotetramethyl-
rosamin-4-yl, tetramethylrhodamin-5-(and 6-)yl, 4,4-
difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-
s-indacene-3-ethyl.
Suitable functionalized fluorescent dye radicals which
can serve as reagents for the preparation of the
compounds of the formula I according to the invention
are described, for example, in "Handbook of Fluorescent
Probes and Research Chemicals, 5th Edition, 1992-1994,
by R.P. Haughland, Molecular Probes, Inc.".
The invention comprises not only the peptides mentioned
but also mixtures and preparations which, in addition
to these compounds according to the invention, also
contain other pharmacological active compounds or
adjuvants which can influence the primary
pharmacological action of the peptides according to the
invention in a desired manner.
The compounds according to the invention and also the
starting substances for their preparation are otherwise
prepared by methods which are known per se and
frequently employed, such as are described in the
literature (e. g. in the standard works such as Houben-
Weyl, Methoden der organischen Chemie (Methods of
Organic Chemistry), Georg-Thieme Verlag, Stuttgart),
namely under reaction conditions which are known and
suitable for the reactions mentioned. Use can also be
made in this case of variants which are known per se.
CA 02379022 2002-O1-11
- 13 -
Preferably, the peptides according to the invention can
be prepared by means of solid-phase synthesis and
subsequent removal and purification, as was described,
for example, by Jonczyk and Meienhofer (Peptides, Proc.
th
8 Am. Pept. Symp., Eds. V. Hruby and D.H. Rich, Pierce
Comp. III, pp. 73-77, 1983, or Angew. Chem. 104, 1992,
375) or according to Merrifield (J. Am. Chem. Soc. 94,
1972, 3102).
The peptides according to the invention can be prepared
on a solid phase (manually or in an automated
synthesizer) in an Fmoc strategy using acid-labile side
protective groups and purified by means of RP-HPLC. The
peak homogeneity can be measured by RP-HPLC and the
substance identity by means of FAB-MS.
Otherwise, the peptides can be prepared by customary
methods of amino acid and peptide synthesis, such as is
known, for example, from Novabiochem - 1999 Catalog &
Peptide Synthesis Handbook of Calbiochem-Novabiochem
GmbH, D-65796 Bad Soden, from numerous standard works
and published patent applications.
Stepwise couplings and fragment condensations can be
utilized. Different N-terminal, C-terminal and side
protective groups can be used, which are preferably
selected to be orthogonally cleavable. Coupling steps
can be carried out using different condensing reagents
such as carbodiimides, carbodiimidazole, those of the
uronium type such as TBTU, mixed anhydride methods, and
acid halide or active ester methods. Activated esters
are expediently formed in situ, for example by addition
of HOBt or N-hydroxysuccinimide.
The cyclization of a linear precursor molecule having
side protective groups can likewise be carried out
using such condensation reactions, as is described, for
example, in DE 43 10 643 or in Houben-Weyl, 1.c.,
Volume 15/II, pages 1 to 806 (1974).
CA 02379022 2002-O1-11
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Different resins and anchor functions can be utilized
in the solid-phase peptide synthesis. Resins can be
based, for example, on polystyrene or polyacrylamide,
anchor functions such as Wang, o-chlorotrityl are
utilizable for the preparation of peptide acids,
aminoxanthenoxy anchors, for example for the
preparation of peptide amides.
Biotinylated or fluorescence-labelled peptides/proteins
can likewise be prepared by standard methods (for
example E.A. Bayer and M. Wilchek in Methods of
Biochemical Analysis Vol. 26 The Use of the Avidin-
Biotin Complex as a Tool in Molecular Biology; and
Handbook of Fluorescent Probes and Research Chemicals,
6th Edition, 1996, by R.P. Haugland, Molecular Probes,
Inc.; or alternatively WO 97/Z47Z6).
Of course, the peptides according to the invention can
also be liberated by solvolysis, in particular
hydrolysis, or by hydrogenolysis of their functional
derivatives. Preferred starting substances for the
solvolysis or hydrogenolysis are those which, instead
of one or more free amino and/or hydroxyl groups,
contain corresponding protected amino and/or hydroxyl
groups, preferably those which, instead of an H atom
which is connected to an N atom, carry an amino
protective group or which, instead of the H atom of a
hydroxyl group, carry a hydroxyl protective group. The
same applies to carboxylic acids which can be protected
by substitution of their -CO-OH hydroxyl function by
means of a protective group, for example as an ester.
The expression "amino protective group" is generally
known and relates to groups which are suitable for
protecting (or blocking) an amino group from chemical
reactions, but which are easily removable after the
desired chemical reaction has been carried out at other
positions in the molecule. The expression "hydroxyl
protective group" is likewise generally known and
CA 02379022 2002-O1-11
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relates to groups which are suitable for protecting a
hydroxyl group from chemical reactions, but which are
easily removable after the desired chemical reaction
has been carried out at other positions in the
molecule. The liberation of the compounds from their
functional derivatives takes place - depending on the
protective group utilized - for example using strong
acids, expediently using TFA or perchloric acid, but
also using other strong inorganic acids such as
hydrochloric acid or sulfuric acid, strong organic
carboxylic acids such as trichloroacetic. acid or
sulfonic acids such as benzene- or p-toluenesulfonic
acid. Hydrogenolytically removable protective groups
(for example CBZ or benzyl) can be removed, for
example, by treatment with hydrogen in the presence of
a catalyst ( for example of a noble metal catalyst such
as palladium, expediently on a support such as carbon).
Typical protective groups for N termini and for side-
position amino groups are Z, BOC, Fmoc, those for C-
termini or the Asp or Glu side chains are O-prim-alkyl
(for example OMe or OEt), 0-tert-alkyl (for example
OBut) or OBenzyl. Z, BOC, NO2, Mtr, Pmc or Pbf, for
example, is suitable for the guanidino function of the
Arg. Alcoholic functions can be protected by benzyl
radicals, tert-alkyl radicals or trityl groups.
The groups BOC, OBut and Mtr can preferably be removed,
for example, using TFA in dichloromethane or using
approximately 3 to 5 N HC1 in dioxane at 15-30°, the
FMOC [sic] group using an approximately 5 to 500
solution of dimethylamine, diethylamine or piperidine
in DMF at 15-30°.
The trityl group is employed, for example, for the
protection of the amino acids histidine, asparagine,
glutamine and cysteine. Removal is carried out,
depending on the desired final product, using TFA/10~
thiophenol, the trityl group being removed from all
CA 02379022 2002-O1-11
- 16 -
amino acids mentioned, when using TFA/anisole or
TFA/thioanisole only the trityl group of His, Asn and
Gln is removed, whereas that on the Cys side chain
remains.
Hydrogenolytically removable protective groups (for
example CBZ or benzyl) can be removed, for example, by
treatment with hydrogen in the presence of a catalyst
(for example of a noble metal catalyst such as
palladium, expediently on a support such as carbon).
Suitable solvents in this case are those indicated
above, in particular, for example, alcohols such as
methanol or ethanol or amides such as DMF. As a rule,
the hydrogenolysis is carried out at temperatures
between approximately 0 and 100° and pressures between
approximately 1 and 200 bar, preferably at 20-30° and
1-10 bar. Hydrogenolysis of the CBZ group takes place
readily, for example, on 5 to 10~ Pd/C in methanol or
using ammonium formate (instead of hydrogen) on Pd/C in
methanol/DMF at 20-30°.
As already mentioned, the peptides according to the
invention include their physiologically acceptable
salts, which can likewise be prepared by standard
methods. Thus, a base of a compound according to the
invention can be converted into the associated acid
addition salt using an acid, for example by reaction of
equivalent amounts of the base and of the acid in an
inert solvent such as ethanol and subsequent
evaporation. Acids which are suitable for this reaction
are in particular those which yield physiologically
acceptable salts. Thus, inorganic acids can be used,
for example sulfuric acid, nitric acid, hydrohalic
acids such as hydrochloric acid or hydrobromic acid,
phosphoric acids such as orthophosphoric acid, sulfamic
acid, furthermore organic acids, in particular
aliphatic, alicyclic, araliphatic, aromatic or hetero-
cyclic mono- or polybasic carboxylic, sulfonic or
sulfuric acids, e.g. formic acid, acetic acid,
CA 02379022 2002-O1-11
- 17 -
propionic acid, pivalic acid, diethylacetic acid,
malonic acid, succinic acid, pimelic acid, fumaric
acid, malefic acid, lactic acid, tartaric acid, malic
acid, citric acid, gluconic acid, ascorbic acid,
nicotinic acid, isonicotinic acid, methane- or
ethanesulfonic acid, ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, naphthalenemono- and
-disulfonic acids and laurylsulfuric acid. Salts with
physiologically unacceptable acids, e.g. picrates, can
be used for the isolation and/or purification of the
compounds according to the invention. ~n the other
hand, an acid of the compounds according to the
invention can be converted into one of its
physiologically acceptable metal or ammonium salts by
reaction with a base. Possible salts in this case are
in particular the sodium, potassium, magnesium, calcium
and ammonium salts, furthermore substituted ammonium
salts, for example the dimethyl-, diethyl- or
diisopropylammonium salts, monoethanol-, diethanol- or
diisopropylammonium salts, cyclohexyl- or
dicyclohexylammonium salts, dibenzylethylenediammonium
salts, furthermore, for example, salts with arginine or
lysine.
The peptide compounds according to the invention can be
employed, as already mentioned, as pharmaceutical
active compounds in human and veterinary medicine, in
particular for the prophylaxis and/or therapy of
disorders in which epithelial cells are involved.
Particularly to be emphasized in this context are
disorders or inflammations or wound-healing processes
of the skin, the respiratory tract organs and the
stomach and intestinal region, thus, for example,
apoplexy, angina pectoris, oncoses, osteolytic diseases
such as osteoporosis, pathological angiogenic diseases
such as, for example, inflammation, fibrosis, in
particular pulmonary fibrosis, opthalmological
diseases, diabetic retinopathy, macular degeneration,
CA 02379022 2002-O1-11
- 18 -
myopia, ocular histoplasmosis, rheumatoid arthritis,
osteoarthritis, rubeotic glaucoma, ulcerative colitis,
Crohn's disease, atherosclerosis, psoriasis, restenosis
after angioplasty, in acute kidney failure, nephritis,
microbial infections and multiple sclerosis.
The invention accordingly relates to peptide compounds
of the formulae defined above and below and in the
claims including their physiologically acceptable salts
as medicaments, diagnostics or reagents.
The invention in particular relates to appropriate
medicaments as inhibitors for the control of disorders
which are indirectly or directly based on expression of
the oc~,~3s integrin receptor, thus in particular in
pathological angiogenic disorders, thromboses, cardiac
infarct, coronary heart disorders, arteriosclerosis,
tumours, osteoporosis, inflammation, infections and for
influencing wound-healing processes.
[lacuna] also relates to appropriate pharmaceutical
preparations, which contain at least one medicament of
the formula I and, if appropriate, vehicles and/or
excipients.
The invention furthermore relates to the use of the
peptide compounds and/or their physiologically
acceptable salts according to the claims and the
description for producing a medicament for the control
of disorders which are based indirectly or directly on
expression of the ocV(36 integrin receptor, thus in
particular in pathological angiogenic disorders,
thromboses, cardiac infarct, coronary heart disorders,
arteriosclerosis, tumours, osteoporosis, inflammation,
infections and for influencing wound-healing processes.
The medicaments according to the invention or
pharmaceutical preparations comprising them can be used
in human or veterinary medicine. Possible vehicles are
organic or inorganic substances which are suitable for
enteral (e.g. oral) or parenteral administration, or
CA 02379022 2002-O1-11
- 19 -
topical application or for administration in the form
of an inhalation spray and do not react with the novel
compounds, for example water, vegetable oils, benzyl
alcohols, alkylene glycols, polyethylene glycols,
glycerol triacetate, gelatin, carbohydrates such as
lactose or starch, magnesium stearate, talc, petroleum
jelly. In particular, tablets, pills, coated tablets,
capsules, powders, granules, syrups, juices or drops
are used for oral administration, suppositories are
used for rectal administration, solutions, preferably
oily or aqueous solutions, furthermore suspensions,
emulsions or implants, are used for parenteral
administration, and ointments, creams or powders are
used for topical application. The novel compounds can
also be lyophilized and the lyophilizates obtained can
be used, for example, for the production of injection
preparations. The preparations indicated can be
sterilized and/or can contain excipients such as
lubricants, preservatives, stabilizers and/or wetting
agents, emulsifiers, salts for influencing the osmotic
pressure, buffer substances, colorants, flavourings
and/or [lacuna] more further active compounds, e.g. one
or more vitamins.
For administration as an inhalation spray, sprays can
be used which contain the active compound either in
dissolved form or suspended in a propellant or
propellant mixture (for example C02 or fluoro
chlorohydrocarbons). The active compound is expediently
used in this case in micronized form, where one or more
additional physiologically tolerable solvents can be
present, e.g. ethanol. Inhalation solutions can be
administered with the aid of customary inhalers.
As a rule, the substances according to the invention
can be administered in analogy to other known,
commercially available peptides (for example described
in US-A-4 472 305), preferably in doses between
approximately 0.05 and 500 mg, in particular between
0.5 and 100 mg, per dose unit. The daily dose is
CA 02379022 2002-O1-11
- 20 -
preferably between approximately 0.01 and 20 mg/kg of
body weight. The specific dose for each patient
depends, however, on all sorts of factors, for example
on the efficacy of the specific compound employed, on
the age, body weight, general state of health, sex, on
the diet, on the time and route of administration, and
on the excretion rate, pharmaceutical combination and
severity of the particular disorder to which the
therapy relates. Parenteral administration is
preferred.
Furthermore, the novel compounds of the formula I can
be used in analytical biology and molecular biology.
The novel compounds of the formula I, where X is a
fluorescent dye radical linked via a -CONH-, -C00-,
-NH-C(=S)-NH-, -NH-C(=0)-NH-, -S02NH- or -NHCO- bond,
can be used as diagnostic markers in the FACS
(Fluorescence Activated Cell Sorter) technique and
fluorescence microscopy.
The use of labelled compounds in fluorescence
microscopy is described, for example, by Y.-L. Wang and
D.L. Taylor in "Fluorescence Microscopy of Living Cells
in Culture, parts A + B, Academic Press, Inc. 1989".
The novel compounds according to the invention can also
be used as integrin ligands for the preparation of
columns for affinity chromatography for the preparation
of integrins in pure form. The complex of an avidin-
derivatized support material, e.g. Sepharose, and the
novel compounds is formed by methods known per se (for
example E.A. Bayer and M. Wilchek in Methods of
Biochemical Analysis Vol 26 The Use of the Avidin-
Biotin Complex as a Tool in Molecular Biology).
Suitable polymeric support materials here are the
polymeric solid phases having preferably hydrophilic
properties and known per se in peptide chemistry, for
example crosslinked polysugars such as cellulose,
CA 02379022 2002-O1-11
- 21 -
Sepharose or Sephadex~, acrylamides, polymers based on
polyethylene glycol or Tentakel polymers.
The invention finally also includes recombinant DNA
sequences which contain sections which code for peptide
regions which have the peptide structural motifs
according to the invention.
DNA of this type can be transferred to cells by
particles, as is described in Ch. Andree et al. Proc.
Natl. Acad. Sci. 91, 12188-12192 (1994), or the
transfer to cells can be increased by other excipients,
such as liposomes (A.I. Aronsohn and J.A. Hughes J.
Drug Targeting, 5, 163-169 (1997)).
The transfer of a DNA of this type could accordingly be
utilized in yeasts, by means of baculoviruses or in
mammalian cells for the production of the peptide
substances of this invention.
If an animal or human body is infected with a
recombinant DNA of this type, the peptides according to
the invention finally themselves formed by the infected
cells can immediately bind to the oc,~,~i6 integrin
receptor, for example of tumour cells, and block it.
Corresponding recombinant DNA, which can be prepared by
known and customary techniques, can, for example,
however also be present in the form of virus DNA which
contains sections which code for the virus coat
protein. By infection of a host organism with
recombinant, preferably non-pathogenic viruses of this
type, host cells which express the integrin ot~,(36 can
preferably be attacked (targeting).
Suitable viruses are, for example, adenoviral types
which have been used a number of times already as
vectors for foreign genes in mammalian cells . A number
of properties make them good candidates for gene
therapy, as can be seen from S.J. Watkins et al. Gene
CA 02379022 2002-O1-11
- 22 -
Therapy 4, 1004-1012 (1997) (see also J. Engelhardt et
al. Hum. Gene Ther. 4, 759-769 (1993)).
As can be found in A. Fasbender et al. J. Clin. Invest.
102, 184-193 (1998), a common problem in gene therapy
by means of viral and non-viral vectors is the limited
efficiency of the gene transfer. Using the additional
ligand sequence for oc~,(36 integrin described above in the
coat protein of the adenoviruses, an improvement in the
transfer, for example, of cystic fibrosis transmembrane
conductance regulator (CFTR) cDNA can be achieved.
In a manner similar to that in the work of T. Tanaka et
al. Cancer Research 58, 3362-3369 (1998), instead of
the DNA for angiostatin the DNA for the sequences of
this invention can also be utilized for cell
transfections by means of retroviral or adenoviral
vectors.
The peptides according to the invention can also be
employed within a liposome complex of lipid/peptide/DNA
for a transfection of cell cultures together with a
liposome complex consisting of lipid/DNA (without
peptide) for use in gene therapy in man. The
preparation of a liposome complex , from
lipid/DNA/peptide is described, for example, in Hart
S.L, et al. 1998: Lipid-Mediated Enhancement of
Transfection by a Non-Viral Integrin-Targeting Vector.
Human Gene Therapy 9, 575-585.
A liposome complex of lipid/peptide/DNA can be
prepared, for example, from the following stock
solutions: 1 [ug/~1 of lipofectin (equimolar mixture of
DOTMA (= N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl
ammonium chloride) and DOPE (dioleyl phosphatidyl
ethanolamine)), 10 ~g/ml of plasmid DNA and 100 ~g/ml
of peptide. For this, both DNA and peptide are
dissolved in cell culture medium.
The liposome complex is prepared by mixing the three
components in a specific weight ratio
(lipid: DNA: peptide, for example 0.75:1:4). Liposomal
CA 02379022 2002-O1-11
- 23 -
DNA complexes for gene therapy in man have already been
described (Caplen N.J. et al. 1995: Liposome-mediated
CFTR gene transfer to the nasal epithelium of patients
with cystic fibrosis Nature Medicine 1, 39-46).
The invention thus also relates to the use of
accordingly modified recombinant DNA of gene-releasing
systems, in particular virus DNA, for the control of
diseases which are based indirectly or directly on
expression of cc~(36 integrin receptors, thus in
particular in pathological angiogenic disorders,
thromboses, cardiac infarct, coronary heart disorders,
arteriosclerosis, tumours, osteoporosis, inflammation,
infections and for influencing wound-healing processes.
Above and below, all temperatures are indicated in °C.
The HPLC analyses (retention time Rt) were carried out
in the following systems:
Column 5 ~..im LichroSpher 60 RP-Select B (250-4) , with a
50-minute gradient from 0 to 80~ 2-propanol in
water/0.3~ trifluoroacetic acid, at 1 ml/min flow and
detection at 215 nm.
Mass spectrometry (MS): EI (electron impact
ionization) M+
FAB (fast atom bombardment)
(M+H)+
Example 1
Preparation and purification of peptides according to
the invention:
In principle, the preparation and purification were
carried out by means of Fmoc strategy with protection
of acid-labile side chains on acid-labile resins using
a commercially obtainable "continuous flow" peptide
synthesizer according to Haubner et al. (J. Am. Chem.
Soc. 118, 1996, 17703).
CA 02379022 2002-O1-11
- 24 -
Cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-Abu)
[EMD 272914]
2.7 g of Fmoc-Abu-OH (Bachem B-1910) was [sic]
suspended in 150 ml of methylene chloride and dissolved
until clear using 10 ml of DMF. 5.6 ml of
diisopropylethylamine were added, the solution was
poured onto 12.0 g of o-chlorotrityl chloride
polystyrene resin (1.14 mmol/g, Bachem D-196512) and
the batch was shaken at room temperature.
After 5 hours, the resin was filtered off with suction
and washed with 300 ml each of DCM/MeOH/DIEA = 17/2/1,
DCM, DMF, DCM and MeOH. After removal of the solvents,
14.55 g of Fmoc-amino acid resin were obtained. A
triplicate Fmoc determination showed, on average, a
loading of 541 [umol/g of Fmoc-Abu-O-oClTrt-resin.
0.7 g of Fmoc-Abu-0-aClTrt-polystyrene resin was
successively subjected to a coupling step in a double-
coupling technique 2 x with 0.30 g each of TBTU,
0.315 ml of ethyldiisopropylamine and Fmoc-amino acid
in 4.1 ml of DMF in a commercial synthesis apparatus
and a typical procedure (Milligen 9050 Pep SynthesizerTM
apparatus and handbook, 1987), for 30 minutes in each
case. Washing steps were carried out in DMF for 10
minutes, cleavage steps in piperidine/DMF (1:4 vol) for
5 minutes, and N-terminal acetylations (capping) were
carried out with acetic anhydride/pyridine/DMF (2:3:15
vol) for 15 minutes.
The amino acids Fmoc-Arg(Pmc), then Fmoc-Leu, then
Fmoc-Ala, then Fmoc-D-Asp(OBut), then Fmoc-Leu, then
Fmoc-Asp(OBut), then Fmoc-Thr(But), then Fmoc-Arg(Pmc)
and finally Fmoc-Abu were used. After cleavage of the
Fmoc protective group from the Fmoc-Abu-Arg(Pmc)-
Thr(But)-Asp(OBut)-Leu-D-Asp(OBut)-Ala-Leu-Arg(Pmc)-
Abu-0-oClTrt-polystyrene resin, it was washed with DMF
and isopropanol and, after drying in vacuo at room
temperature, 0.9 g of Abu-Arg(Pmc)-Thr(But)-Asp(OBut)-
CA 02379022 2002-O1-11
- 25 -
Leu-D-Asp(OBut)-Ala-Leu-Arg(Pmc)-Abu-0-oClTrt-
polystyrene resin was obtained.
By treatment of this peptidyl resin with 20 ml of
trifluoroethanol/dichloromethane/acetic acid (2:6:2
vol) for 2 hours at room temperature, filtration,
concentration in vacuo and trituration with diethyl
ether, 0.19 g of the side chain-protected peptide
Abu-Arg(Pmc)-Thr(But)-Asp(OBut)-Leu-D-Asp(OBut)-Ala-
Leu-Arg(Pmc)-Abu-OH was obtained.
By dropwise addition, of a solution of this product in
DMF (100 mg of peptide/15 ml of DMF) to a stirred
solution of TBTU/HOBt/DIPEA (10:10:11 equivalents) in
50 ml of DMF/100 mg of peptide in the course of
30 minutes and further stirring for 1 hour, cyclization
was achieved. After concentration and precipitation
with water, 0.15 g of crude cyclo(Arg(Pmc)-Thr(But)-
Asp(OBut)-Leu-D-Asp(OBut)-Ala-Leu-Arg(Pmc)-Abu-Abu) was
obtained. After treatment with trifluoroacetic
acid/water/TIS (94:3:3 vol) for 2 hours at room
temperature, concentration in vacuo and trituration
with diethyl ether, a precipitate of 85 mg of
cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-Abu) was
obtained.
Purification of the product was carried out by RP-HPLC
on Lichrosorb RP 18 (250 - 25, 7 wm, Merck KGaA) in
0.3~ TFA using a gradient of 4~ to 24~ 2-propanol over
one hour at 10 ml/min and assessment of the eluate by
means of a UV flow-through photometer at 215 and
254 nm. 51 mg of product were obtained, FAB 1067; Rt
[sic] 19Ø
The following products were prepared analogously:
Code Sequence MW FAB Rt
(E~) (g/mol) [sic]
271312 cyclo(RTDLDSLR) 957.1 958 15.10
271578 cyclo(RTDLdSLR) 957.1 957 17.51
CA 02379022 2002-O1-11
- 26 -
Code S equence M W F AB R t
(E~) (g/mol) Lsic]
271579 cyclo(RTDLdALR) 941.1 941 16.64
271586 cyclo(RGDLDSLR) 913.0 913 17.95
271587 cyclo(RGDLdSLR) 913.0 913 17.24
271588 cyclo(RGDLdALR) 897.0 897 19.60
271589 cyclo(RGDLdGLR) 882.9 883 16.68
271590 cyclo(RGDLd-~3-Ala-LR) 897.0 897 15.47
271591 cyclo(RGDLdALRG) 954.1 954 16.79
271592 cyclo(RGDLdALRGG) 1011.1 1011 17.83
271593 cyclo(RGDLdALRGGG) 1068.2 1068 17.79
271594 cyclo(RGDLdALRGGGGGG) 1239.3 1239 17.30
272914 cyclo(RGDLdALR-Abu-Abu) 1067.2 1067 19.00
272966 cyclo(RTDLdALRGGG) 1112.2 1113 17.14
272967 cyclo(RTDLdGLRGGG) 1098.2 1099 19.00
272968 cyclo(RTDLDALRGGG) 1112.2 1113 17.39
272969 cyclo(RTDLDGLRGGG) 1098.2 1099 16.88
272970 cyclo(RGDLDALRG) 954.1 955 17.32
272971 cyclo(RaDLdALRGGG) 1082.2 1083 18.26
272972 cyclo(RGDLaALRGGG) 1082.2 1083 18.23
272958 cyclo(RGDLdALR-Aha-Aha) 1123.3 1123 19.38
272959 cyclo(RGDLdALR-Ana) 1010.2 1010 20.63
272960 cyclo(RGDLdALR-Aee) 1042.2 1042 18.92
272961 cyclo(RGDLd.ALRGGGG) 1125.2 1125 17.56
272962 cyclo(RGDLdALRGGGGG) 1182.3 1182 17.52
272963 cyclo(RGDLdGLRGGG) 1054.1 1054 15.59
272964 cyclo(RGDLDALRGGG) 1068.2 1069 16.84
272965 cyclo(RGDLDGLRGGG) 1054.1 1055 16.03
273028 cyclo(RTDLdALR-Aha) 1072.2 n.d. 19.30
273033 cyclo(RTDLdALR-Abu) 1044.2 n.d. 16.72
273035 cyclo(RGDLdALR-Abu) 1000.1 n.d. 16.96
273038 cyclo(RTDLdALR-Aha-Aha) 1185.4 n.d. 20.46
273040 cyclo(RTDLdALR-Abu-Abu) 1111.3 n.d. 18.75
X 2 TFA
304219 cyclo(RTDLdALR-(3Ala) 1012.1 1012 19.4
304218 cyclo(RGDLdALR-~iAla) 968.1 968 18.7
329400 cyclo(RGDLdALR) 811.9 812 21.15
CA 02379022 2002-O1-11
- 27 -
Code Sequence MW FAB Rt
(E~) (g/mol) [sic]
329412 cyclo(RTDLdALR-Abu-Abu) 1111.3 1112 20.31
329402 cyclo(RGDLdALAGGG) 983.1 984 20.48
329399 cyclo(NMeArg- 1038.2 1039 19.66
GDLaALRGGG)
329398 cyclo(R-NMeGly- 1038.2 1039 19.76
DLaALRGGG)
326397 cyclo(RGD-NMeLeu- 1038.2 1039 20.22
aALRGGG)
329396 cyclo(RGDL-[D-NMeAla]- 1038'.2 1039 20.55
ALRGGG)
329395 cyclo(RGDLa-NMeAla- 1038.2 1039 21.35
LRGGG)
329394 cyclo(RGDLaA-NMeLeu- 1038.2 1039 18.87
RGGG)
329393 cyclo(RGDLaAL-NMeArg- 1038.2 1039 20.64
GGG)
cyclo(RGDLdAAR)
cyclo(RGDLdAARGGG)
Nomenclature for amino acids according to Eur. J.
Biochem. 138, 9-37 (1984)
lower case letter = D-amino acid
n.d. - not determined
Example 2:
a~/~6 / fibronectin receptor binding test:
The prepared peptides according to the invention were
bonded to the immobilized a"(36 receptor together with
competitively acting fibronectin in solution and the Q
value was determined as a measure of the selectivity of
the binding of the peptide to be tested to a~,(36. The Q
value is calculated here from the quotient of the ICSo
values of test peptide and a standard. The standard
used was the linear Ac-RTDLDSLR-NH2 (Code EMD 271293)
(ref./patent cf. Pytela et al. Science 231, 1559,
CA 02379022 2002-O1-11
- 28 -
(1986) ) . The binding test was carried out in detail as
follows:
The immobilization of soluble oc",(36 receptor on
microtitre plates was carried out by dilution of the
protein solution in TBS++ and subsequent incubation
overnight at 4°C (100 ~.l;hollow). Non-specific binding
sites were blocked by incubation (2 h, 37°C) with 30
(w/v) BSA in TBS++ (200 ~1/hollow). Excess BSA was
removed by washing three times with TBSA++. Peptides
were diluted serially (1:10) in TBSA++ and incubated
with the immobilized integrin (50 ~1 of peptide + 50 ~.1
of ligand per hollow; 2 h; 37°C) together with
biotinylated fibronectin (2 ~.g/ml). Unbound fibronectin
and peptides were removed by washing three times with
TBSA++. The bound fibronectin was detected by
incubation (1 h; 37°C) with an alkaline phosphatase-
coupled anti-biotin antibody (Biorad) (1:20,000 in
TBSA++; 100 ~l/hollow). After washing three times with
TBSA++, colorimetric detection was carried out by
incubation (10-15 min; 25°C, in the dark) with
substrate solution (5 mg of nitrophenyl phosphate, 1 ml
of ethanolamine, 4 ml of H20; 100 ~1/hollow). The enzyme
reaction was stopped by addition of 0.4 M NaOH (100
~.l/hollow). The colour intensity was determined at 405
nm in an ELISA measuring apparatus and made equal to
the zero value. Hollows which were not coated with
receptor served as a zero value. Ac-RTDLDSLR-NH2 was
employed as a standard. The ICSO values for the peptides
tested were read off from a graph and from this,
together with the ICSo value of the standard peptide,
the Q value of the peptide according to the invention
was determined.
Q value = ICSO test peptide / ICSO standard
Q values were calculated as means from repeat
experiments.
The results of the test described are summarized in the
Table 1 which follows:
CA 02379022 2002-O1-11
- 29 -
Table 1
Results of the oc,~,(36 / fibronectin receptor binding test
Code (E1~) Sequence Q value = ICSo
test peptide /
ICSO EMD 271293
271293 Ac-RTDLDSLR-NH2 1.00 (=75 nM)
271586 cyclo(RGDLDSLR) 26
271587 cyclo(RGDLdSLR) 88
271588 cyclo(RGDLdALR) 47
271589 cyclo(RGDLdGLR) 19
272970 cyclo(RGDLDALRG) 6.7
272964 cyclo(RGDLDALRGGG) 0.037
272965 cyclo(RGDLDGLRGGG) 0.16
272972 cyclo(RGDLaALRGGG) 0.05
271593 cyclo(RGDLdALRGGG) 0.03
272963 cyclo(RGDLdGLRGGG) 0.084
271590 cyclo(RGDLd-~iAla-LR) 233
271591 cyclo(RGDLdALRG) 2.1
271592 cyclo(RGDLdALRGG) 0.05
271594 cyclo(RGDLd.ALRGGGGGG) 0.05
272914 cyclo(RGDLdALR-Abu-Abu) 0.03
272958 cyclo(RGDLdALR-Aha-Aha) 0.036
272959 cyclo(RGDLdALR-Aha) 0.036
272960 cyclo(RGDLd.ALR-Aee) 0.038
272961 cyclo(RGDLdALRGGGG) 0.033
272962 cyclo(RGDLdALRGGGGG) 0.046
273035 cyclo(RGDLdA~R-Abu) 0.028
271312 cyclo(RTDLDSLR) 8
271578 cyclo(RTDLdSLR) 104
271579 cyclo(RTDLdALR) 12
272966 cyclo(RTDLdALR-GGG) 0.05
272967 cyclo(RTDLdGLR-GGG) 0.15
272968 cyclo(RTDLDALR-GGG) 0.14
272969 cyclo(RTDLDGLR-GGG) 0.18
CA 02379022 2002-O1-11
- 30 -
Code (EMD) Sequence Q value = ICSo
test peptide /
ICSO EMD 271293
273028 cyclo(RTDLdALR-Aha) 0.015
273033 cyclo(RTDLd.ALR-Abu) 0.043
273038 cyclo(RTDLdALR-Aha-Aha) 0.015
273040 cyclo(RTDLdALR-Abu-Abu) 0.014
272971 cyclo(RaDLdALR-GGG) 8.2
The following examples relate to pharmaceutical
preparations:
Example A: injection vials
A solution of 100 g of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-
Leu-Arg-Abu-Abu) and 5 g of disodium hydrogenphosphate
is adjusted to pH 6.5 in 3 1 of double-distilled water
using 2 N hydrochloric acid, sterile-filtered,
dispensed into injection vials, lyophilized under
sterile conditions and aseptically sealed. Each
injection vial contains 5 mg of active compound.
Example B: suppositories
A mixture of 20 g of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-
Leu-Arg-Abu-Abu) is fused with 100 g of soya lecithin
and 1400 g of cocoa butter, poured into moulds and
allowed to cool. Each suppository contains 20 mg of
active compound.
Example C: solution
A solution is prepared from 1 g of cyclo(Arg-Thr-Asp-
Leu-D-Asp-Ala-Leu-Arg-Abu-Abu), 9.38 g of NaHzP04
2 H20 , 2 8 . 4 8 g o f Na2HP04 ~ 12 H20 and 0 . 1 g o f
benzalkonium chloride in 940 ml of double-distilled
water. The mixture is adjusted to pH 6.8, made up to
CA 02379022 2002-O1-11
- 31 -
1 1 and sterilized by irradiation. This solution can be
used in the form of eye drops.
Example D: ointment
500 mg of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-
Abu) are mixed with 99.5 g of petroleum jelly under
aseptic conditions.
Example E: tablets
A mixture of 1 kg of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-
Leu-Arg-Abu-Abu), 4 kg of lactose, 1.2 kg of potato
starch, 0.2 kg of talc and 0.1 kg of magnesium stearate
is compressed in a customary manner to give tablets in
such a way that each tablet contains 10 mg of active
compound.
Example F: coated tablets
Analogously to Example E, tablets are pressed and are
then coated in a customary manner with a coating of
sucrose, potato starch, talc, tragacanth and colorant.
Example G: capsules
2 kg of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu
Abu) are dispensed into hard gelatin capsules in a
customary manner such that each capsule contains 20 mg
of the active compound.
Example H: ampoules
A solution of 1 kg of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-
Leu-Arg-Abu-Abu) in 60 1 of double-distilled water is
sterile-filtered, dispensed into ampoules, lyophilized
under sterile conditions and aseptically sealed. Each
ampoule contains 10 mg of active compound.
CA 02379022 2002-O1-11
- 32 -
Example I: inhalation spray
14 g of cyclo(Arg-Thr-Asp-Leu-D-Asp-Ala-Leu-Arg-Abu-
Abu) are dissolved in 10 1 of isotonic NaCl solution
and the solution is dispensed into commercially
available spray containers having a pump mechanism. The
solution can be sprayed into the mouth or nose. One
puff of spray (approximately 0.1 ml) corresponds to a
dose of approximately 0.14 mg.
